RT2 PreAMP Pathway Primer Mixes

For amplification of cDNA templates prior to analysis on RT² Profiler PCR Arrays

Features

  • Primer mixes for human, mouse, and rat
  • Minimal hands-on time
  • Robust performance on small samples

Useful Resources

RT2 PreAMP Pathway Primer Mix

Cat. No. / ID: 330241

RT2 Nano PreAMP Primer Mix
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The RT2 PreAMP Pathway Primer Mixes is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

Product Details

RT² PreAMP cDNA Synthesis Kit and RT² PreAMP Pathway Primer Mixes are a novel technology enabling gene expression analysis using as little as 1 ng total RNA. A  proprietary amplification process increases the amount of cDNA for subsequent PCR array analysis. Starting sample types include fine needle biopsy and laser captured microdissection samples, stem cell clusters or embryoid bodies, and fluorescence-activated cell sorter (FACS) generated cell populations. Primer mixes are available for all cataloged human, mouse, and rat RT2 Profiler PCR Arrays.

Performance

Increased positive call rate and unbiased amplification for unbiased results

More genes are detected after preamplification using the RT² PreAMP Pathway Primer Mix (see figure " Increased positive call rate").

The amplification process is unbiased, resulting in highly comparable ΔCT values between preamplified and unamplified cDNA, as evaluated by regression analysis (see figure " Unbiased amplification process").

There is a high correlation of gene expression fold change results between preamplified and unamplified samples (see figure " Faithfully amplified biology").

See figures

Principle

RT² PreAMP technology utilizes multiplex tandem PCR to preamplify gene-specific cDNA with minimal bias. Following standard reverse transcription, an RT2 PreAMP Pathway Primer Mix is used to amplify templates and enable gene expression analysis on up to 4 different  RT2 Profiler PCR Arrays (see flowchart " Simple template amplification and gene-expression analysis").
See figures

Procedure

First strand cDNA is first synthesized from up to 12 different RNA samples using the RT2 PreAMP cDNA Synthesis Kit. The cDNA is then preamplified for a pathway-specific set of genes. Each first strand cDNA synthesis reaction can be amplified using up to 4 different RT2 PreAMP Pathway Primer Mixes, allowing gene expression analysis on up to 4 different RT2 Profiler PCR Arrays. The Side Reaction Reducer included in the RT2 PreAMP cDNA Synthesis Kit eliminates the residual primers from preamplification, enabling accurate detection on RT2 Profiler PCR Arrays. To complete the PCR array procedure, preamplified templates are mixed with an instrument-specific, ready-to-use RT² SYBR® Green Mastermix. 

Applications

cDNA templates generated using the RT2 PreAMP cDNA Synthesis Kit in conjunction with RT2 PreAMP Pathway Primer Mixes are ready for gene expression profiling using RT² Profiler PCR Arrays.

Supporting data and figures

Resources

Brochures & Guides (1)
Simultaneously profile mRNA, miRNA and lncRNA using a simple, complete workflow

FAQ

Is it good to pool multiple RNA replicates to detect expression changes that are consistently reproducible?
With the additional RT2 PreAMP methodology, only 1 ng of RNA is now needed for PCR Array analysis. Pooling RNA from different sources should only be done when there is not enough sample. We recommend running biological replicates.
FAQ ID -2663
How do I create a workspace that is free of DNA contamination, prior to carrying out a qPCR experiment?

Any DNA contamination will artificially inflate the SYBR Green signal, yielding skewed gene expression profiles and false-positive signals. The most common source of DNA contamination is from PCR products generated during previous experiments. Such contamination is most often due to the improper disposal of tubes, tips, and gels that previously came into contact with PCR products. Additionally, PCR products may also contaminate pipettors, racks, work pads, and commonly used reagents such as water and buffers. To minimize the risk of contaminating your experiment with extraneous DNA, the following steps should be taken:

 

  • Remove a single aliquot of water from your PCR-grade stock, sufficient to complete the experiment. This minimizes the number of times that the stock container is opened, thereby minimizing contamination risks.
  • Use only fresh PCR-grade reagents and disposable labware.
  • Treat any labware (tubes, tips, and tip boxes) used in PCR with 10% bleach, before discarding.
  • Maintain a dedicated workspace for PCR setup (perhaps a PCR-only hood), away from areas of the lab where post-PCR work is done, such as running gels, enzyme digestions, and cloning.
  • Change the lab bench pads/papers often and decontaminate lab benches and labware (racks, pipettors, etc.) before each use by washing with 10% bleach, and/or exposing to UV light for at least 10 minutes. This serves to degrade and/or inactivate contaminating DNA.
  • Before, during, and after the experiment, minimize the opening and closing of any tubes or plates used during the experiment.  
FAQ ID -2654